U.S. patent application number 09/912448 was filed with the patent office on 2003-06-19 for synergistic combinations of natural of compounds that control decay of fruits and vegetables and reduce contamination by foodborne human pathogens.
Invention is credited to El Ghaouth, Ahmed, Wilson, Charles L., Wisniewski, Michael E..
Application Number | 20030113421 09/912448 |
Document ID | / |
Family ID | 25431938 |
Filed Date | 2003-06-19 |
United States Patent
Application |
20030113421 |
Kind Code |
A1 |
Wilson, Charles L. ; et
al. |
June 19, 2003 |
Synergistic combinations of natural of compounds that control decay
of fruits and vegetables and reduce contamination by foodborne
human pathogens
Abstract
Plant essential oils and chitosan salts were tested individually
and in combination against a postharvest pathogen Botrytis cinerea
and two foodborne human pathogens Escherichia coli and Listeria
monocytogenes. Four essential oils (cinnamon, allspice, savory, red
thyme) in combination with two chitosan salts (chitosan sorbate and
chitosan propionate) demonstrated synergistic antimicrobial
activity against all three organisms. The synergistic combinations
of essential oils and chitosan salts also demonstrated an eradicant
activity against E. coli on apple disks previously inoculated with
the organism. Synergistic combinations of essential oils and
chitosan salts hold promise of giving superior control of both
postharvest decay organisms and foodborne human pathogens.
Inventors: |
Wilson, Charles L.;
(Shepherdstown, WV) ; El Ghaouth, Ahmed;
(Frederick, MD) ; Wisniewski, Michael E.;
(Shepherdstown, WV) |
Correspondence
Address: |
USDA, ARS, OTT
5601 SUNNYSIDE AVE
RM 4-1159
BELTSVILLE
MD
20705-5131
US
|
Family ID: |
25431938 |
Appl. No.: |
09/912448 |
Filed: |
July 26, 2001 |
Current U.S.
Class: |
426/326 |
Current CPC
Class: |
A23B 7/154 20130101;
A01N 65/22 20130101; A01N 65/00 20130101; A01N 65/00 20130101; A01N
65/28 20130101; A23L 3/3472 20130101; A01N 43/16 20130101; A23L
3/3544 20130101; A01N 65/24 20130101; A01N 65/24 20130101; A01N
65/08 20130101; A23L 3/3526 20130101; A01N 65/28 20130101; A01N
65/08 20130101; A01N 65/22 20130101; A01N 43/16 20130101; A01N
65/22 20130101; A01N 65/06 20130101; A01N 65/06 20130101; A01N
65/08 20130101; A01N 43/16 20130101; A01N 65/24 20130101; A01N
65/00 20130101; A01N 65/28 20130101; A01N 65/08 20130101; A01N
65/28 20130101; A01N 65/06 20130101; A01N 65/08 20130101; A01N
65/06 20130101; A01N 65/22 20130101; A01N 43/16 20130101; A01N
65/28 20130101; A01N 43/16 20130101; A01N 43/16 20130101; A01N
65/24 20130101; A01N 65/06 20130101; A01N 65/22 20130101; A01N
65/08 20130101; A01N 65/28 20130101 |
Class at
Publication: |
426/326 |
International
Class: |
A23K 001/00 |
Claims
We claim:
1. A composition having antibacterial and antifungal properties
comprising a chitosan salt and an essential oil.
2. A composition having antibacterial and antifungal properties
comprising a chitosan salt and an essential oil in a
synergistically effective amount.
3. A composition according to claim 1 wherein said composition
further comprises a surface-active agent.
4. A composition according to claim 1, wherein said essential oil
is selected from the group consisting of cinnamon, savory, red
thyme, allspice, bay, birch, cloves, carvacrol, and hinokitiol and
mixtures thereof.
5. A composition according to claim 1 wherein said composition
further comprises an additional antimicrobial ingredient.
6. A composition according to claim 1 wherein said composition
further comprises at least an optional ingredient selected from the
group consisting of protective colloids, adhesives, binding agents,
chelating agents, thickening agents, thixotropic agents,
penetrating agents, stabilizing agents, sequestering agents,
anti-foam agents, antioxidants, natural or synthetic seasonings
and/or flavors, dyes and/or colorants, vitamins, minerals,
nutrients, enzymes, insecticides, deodorants, and mixtures
thereof.
7. A wipe impregnated with the antibacterial and antifungal
composition according to claim 1.
8. A composition according to claim 1, packaged in a spray
dispenser.
9. A packaging material impregnated with the composition of claim
1.
10. A method for treating a surface of a food product wherein a
composition according to claim 1 is applied onto said surface.
11. A method for protecting food products from bacterial and fungal
infection by contacting the surface of a fruit or vegetable with
the composition according to claim 1.
12. A method for protecting an exposed fresh fruit or an exposed
fresh vegetable from postharvest and foodborne pathogens, the
method comprising: a) applying the composition of claim 1 to the
surface of a fresh fruit or fresh vegetable; and b) obtaining a
fresh fruit or fresh vegetable that is protected from postharvest
and foodborne pathogens.
13. The method according to claim 12 wherein said composition is
directly applied to said food surface.
14. The method according to claim 13 wherein said composition is
directly applied to said food surface by means of being included in
a marinade, breading, seasoning rub, glaze or colorant mixture
which is applied to said food surface.
15. The method according to claim 12 wherein said composition is
applied to food packaging material which is thereafter brought into
contact with said food surface.
16. A method for inhibiting microbial growth or toxin production in
foods contaminated with bacteria and fungi by contacting the
surface of a fruit or vegetable with the composition according to
claim 1.
17. A method for reducing the overall bacterial and fungal content
of a food product by applying to the surface of said product an
effective amount of the composition of claim 1.
18. The method according to claim 17 wherein said food product is a
fruit, vegetable, or edible nut.
19. A food product having reduced bacteria or fungi as a result of
having applied thereon to its surface a composition which
demonstrates efficacy against bacteria or fungi or both, comprising
the composition of claim 1.
20. An exposed fresh fruit or vegetable or a mixture of exposed
fresh fruits and vegetables, comprising: cut pieces of fruit or
vegetable; and a coating on the exposed surfaces of the fruit or
vegetable, the coating comprising the composition of claim 1.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] This invention relates to synergistic combinations of
natural antimicrobial compounds that are effective against
postharvest and foodborne human pathogens.
[0003] 2. Description of the Relevant Art
[0004] Postharvest decay and contamination of fruits and vegetables
with foodborne pathogens have been and continue to be of major
concern to the fruit and vegetable industry. Conservative estimates
place U.S. and Canadian losses of fruits and vegetables from
postharvest decay at around 25% of the harvested crops. This
problem has been further compounded by the risk of contamination of
fresh and processed fruits and vegetables with foodborne pathogens.
Several pathogenic bacteria such as Salmonella spp, Listeria
monocytogenes, Clostridium botulinum, and Escherichia coli 0157:H7
have been shown to occur at base levels on the outer surfaces of a
wide variety of harvested commodities (1988. Microorganisms in
Foods: Application of the Hazard Analysis Critical Control Point
(HACCP) System to Insure Microbiological Safety and Quality,
Silliker et al., Eds. Blackwell Scientific Publications, Oxford,
England). Recent outbreaks of foodborne illness associated with
consumption of fresh horticultural products and non-pasteurized
fruit juices have weakened consumers confidence in the
wholesomeness of fresh produce (Fairchild et al. 1990. The Packer
33: 1-7; Schwartz et al. 1995. The Packer 27: 6; Wells et al. 1997.
Plant Dis. 81: 867-872; Parish et al. 1998. J. Food Protection 61:
280-284).
[0005] Presently, chlorinated washes in conjunction with proper
refrigeration, stringent sanitation, and synthetic fungicides are
the primary means of controlling foodborne pathogens and
postharvest decay. However, the carcinogenicity of trihalomethanes
and the possible regulatory restriction of chlorine present major
challenges for the fresh produce industry to find safe
alternatives. Similar public concern has been raised regarding
fungicide safety. As a result, a number of key postharvest
fungicides have been recently banned or are undergoing critical
re-registration. In addition, some of the fungicides registered for
postharvest use, particularly benzimidazole, are becoming
ineffective because of the development of fungicide-resistant
strains of postharvest pathogens (Spotts et al. 1986. Plant Dis.
70: 106-108; Eckert, J. W. 1991. In: Role of Chemical Fungicides
and Biological Agents in Postharvest Disease Control. Proceedings
of the Workshop on Biological Control of Postharvest Diseases of
Fruits and Vegetables, Shepherdstown, W. Va., USA, Sep. 12-14,
1990, U.S.D.A. and A.R.S. Publication Vol. 92, page 310.). Thus, it
has become apparent that new, safe methodologies are needed to
reduce both decay and contamination of our food supply by foodborne
human pathogens.
[0006] The use of natural plant- and animal-derived antimicrobials,
i.e., compounds that are antibacterial and antifungal, as
alternatives for the control of foodborne human and plant pathogens
provides an attractive means of attacking problems resulting from
the contamination of our food with microorganisms. A variety of
natural plant compounds including spices, herbs, essential oils,
and volatile substances have been shown to suppress the growth of
food-poisoning bacteria (Bowles et al. 1993. J. Food Protection 56:
795-800; Deans et al. 1987. Int. J. Food Microbiol. 5: 165-180;
Aktug et al. 1986. Int. J. Food Microbiol. 3: 349-353).
[0007] In vitro inhibition of the growth of major postharvest
pathogens and the reduction of fruit decay was also observed with
several essential oils, volatile substances, and plant extracts
(Wilson et al. 1987. Plant Dis. 71: 316-319; Wilson et al. 1997.
Plant Dis. 81: 204-210; Pesis et al. 1993. J. Plant Physiol. 142:
717-721; Sholberg et al. 1991. J. Canad. Inst. Food Sci. Tech. 2:
273-276; Mattheis et al. 1993. Plant Dis. 77: 810-814; Vaugh et al.
1993. J. Food Sci. 58: 793-796). Also, the inhibition of the growth
of foodborne pathogens has been reported with bacteriocins (Fowler
et al. 1990. Antibiotics-nisin. In: Food Preservatives, Russel and
Gould, Eds. AVI Publishing, New York; Motlagh, A. 1991. Ph.D.
Thesis, Univ. Wyoming, Laramie, Wyo.; 1992. Food Biopreservatives
of Microbial Origin, Ray and Daeschel, Eds. CRC Press, New York),
with organic acids (Ray, B. 1992. Diacetyl of Lactic Bacteria as a
Food Biopreservative. In: Food Biopreservatives of Microbial
Origin, supra; Arora et al. 1991. Handbook of Applied Mycology Vol.
3. Marcel Dekker, Inc., New York. 621 pages; Al Zaemey et al. 1993.
Mycolog. Res. 97: 1463-1468; Sholberg et al. 1995. Hort. Sci. 30:
1271-1275), and with chitosan (Hadwiger et al. 1980. Plant Physiol.
66: 205-211; El Ghaouth et al. 1992. Phytopath. 82: 398-402). Some
of these compounds (bacteriocins and organic acids) are also used
commercially to control food spoilage. Most current available data
provide only fragmented information on the effectiveness of
combinations of naturally-occurring antimicrobial compounds and on
their effect on both postharvest and foodborne pathogens.
Development of synergistic combinations of natural compounds can
add a new dimension to their use as food preservatives, enhancing
their effectiveness for stability, low toxicity, availability, and
broad utility.
SUMMARY OF THE INVENTION
[0008] We have discovered naturally-occurring compounds that are
both antifungal and bactericidal and combinations of particular
natural compounds that can be used synergistically to control both
major postharvest pathogens and foodborne pathogens.
[0009] In accordance with this discovery, it is an object of the
invention to provide a composition of natural compounds that act
synergistically and are effective against postharvest pathogens and
foodborne pathogens found on fruits and vegetables.
[0010] It is a further object of the present invention to provide a
method for protecting fruits and vegetables from postharvest
pathogens and foodborne pathogens found on fruit and vegetables by
applying to the surface of fruits and vegetables a composition of
natural compounds that act synergistically and are effective
against bacteria and/or fungi.
[0011] It is a still further object of the present invention to
provide a method for reducing the effects of the overall microbial
content of a food product by applying to the surface of fruits and
vegetables a composition of natural compounds that act
synergistically and are effective for eradicating or inhibiting
growth and toxin production of bacteria and fungi found on fruits
and vegetables.
[0012] An additional object of the present invention is to provide
a fruit or vegetable food product having reduced levels of
bacterial and/or fungal postharvest pathogens and foodborne
pathogens.
[0013] Other objects and advantages of the invention will become
readily apparent from the following description.
DETAILED DESCRIPTION OF THE INVENTION
[0014] The present invention provides combinations of chitosan
salts and essential oils that act synergistically both to protect
food products from bacterial and fungal contamination and to
eradicate or at least inhibit growth and toxin production in foods
contaminated with bacteria and fungi. The present invention relates
to effective, inexpensive, and environmentally appropriate
compositions and methods for controlling postharvest pathogens and
foodborne pathogens, as for example, enterotoxigenic bacteria such
as E. coli and L. monocytogenes, on fruits and vegetables.
[0015] Food product here refers to a fruit or a vegetable or part
of a fruit or vegetable that can be infected or contaminated by
postharvest pathogens and foodborne pathogens. The term "food
product" encompasses "exposed fresh fruit" and "exposed fresh
vegetable" which in its broadest sense includes the tissue normally
covered by the skin of the fruit or vegetable which is exposed when
the fruit or vegetable is peeled, cut, segmented or otherwise
exposed. The tissue is fresh or raw and is preferably in the form
of cut or segmented pieces which have not been heat sterilized or
blanched. Generally, one or more of any type of fresh vegetable,
fruit or nut, for example, may be treated with the present
invention. Suitable examples of fruit include apples, apricots,
avocado, bananas, blackberries, blueberries, cherries, cranberries,
custard apples, dates, durian, figs, grapefruit, grapes, jack
fruit, kiwi fruit, lemons, limes, lychee, mandarins, mangosteen,
mangoes, melons, nashi, nectarines, oranges, papaya or paw paw,
passionfruit, peaches, pears, pineapple, plums, pomegranates,
pomelo, raspberries, rhubarb, star fruit, strawberries, tamarillo,
and tangerines of any maturity. Any edible nut is also included.
Suitable non-limitative examples of vegetables include: potatoes,
corn, tomatoes, onions, herbs, squash, beans, peppers, okra,
turnips, broccoli, cauliflower, cabbage, carrots, brussels,
sprouts, zucchini, radishes, celery, lettuce, and even prepared
mixed vegetable salads. Moreover, any fresh vegetable, fruit or nut
may be treated with the present invention, whether grown in the
ground or grown hydroponically.
[0016] As used herein, "foodborne pathogen" refers to a bacterium
or a fungus capable of contaminating a fruit or a vegetable and
causing disease to humans or animals ingesting said fruit or
vegetable.
[0017] As used herein, "postharvest pathogen" refers to a bacterium
or a fungus capable of infecting a fruit or a vegetable and thereby
causing postharvest decay.
[0018] As used herein, the term "synergism" is intended to include
both an increased spectrum of activity (i.e., greater activity
against a broad spectrum of microorganisms), and/or increased
efficacy (i.e., greater activity against specific organisms than
that predicted by use of either agent alone). The increased
antimicrobial and antifungal activity of the synergistic
combination permits the use of smaller amounts of each agent
thereby decreasing costs and minimizing other problems, e.g.,
toxicity, solubility, availability. Effectiveness against a broad
spectrum of microorganisms broadens the utility of the synergistic
product based on its effectiveness in environments containing many
and diverse microorganisms which must be controlled.
[0019] Chitosan is a semisynthetic derivative of chitin produced by
the deacetylation of the nitrogen thereof so as to produce the
ammonium salt. Chitosan has been shown to have some mild antifungal
activity with regard to particular fungal species; see for example,
Hadwiger et al., supra; El Ghaouth et al. 1994. Phytopath. 84:
313-320; El Ghaouth et al., 1992, supra; Allan et al. 1979. Exp.
Mycology 3: 285-287; Stossel et al. 1984. Phytopath. 11: 82-90;
Kendra et al. 1984. Exp. Mycology 8: 276-281, and Ben-Shalom et al.
1999. U.S. Pat. No. 5,965,545.
[0020] The compositions of the invention comprise combinations of
chitosan salts and essential oils that act synergistically both to
protect food products from bacteria and fungi and to eradicate or
inhibit decay and toxin production in foods contaminated with
bacteria and fungi. Particular combinations can be screened in
vitro in culture medium prior to testing on food products. Culture
medium is inoculated with suspensions of bacteria or fungal spores.
Chitosan salts, essential oils, or combinations of chitosan salts
and essential oils are (1) added to the culture medium prior to
inoculation to determine protective effects or (2) added after
inoculation to determine inhibitory or eradicant effects.
[0021] Generally, the compositions according to the invention
usually contain in addition to the active material (chitosan salt
and essential oil), one or more solid or liquid vehicles and,
optionally, one or more surface-active agents. The solid or liquid
vehicles and/or surface-active agents utilized in the compositions
of the invention must be acceptable in agriculture; inert and
conventional vehicles and conventional surface-active agents can be
used. The compositions according to the invention are
pharmaceutically-acceptable, i.e., the compositions or components
are suitable for use in contact with human tissue without undue
toxicity, incompatibility, instability, allergic response, and the
like. These compositions cover not only compositions that are ready
to be applied to the fruits and vegetables, as for example by means
of a suitable device, such as a spray device, but also commercial
concentrated compositions which have to be diluted before
application to the food product.
[0022] In the present account, the term "vehicle" denotes a natural
or synthetic, organic or inorganic material with which the active
material is combined to facilitate its application on the food
product. This vehicle is thus generally inert and it must be
agriculturally and pharmaceutically acceptable. The vehicle can be
solid as for example, clays, natural or synthetic silicates,
resins, and waxes or the vehicle can be liquid, such as water,
alcohols, propylene glycol, a vegetable oil or like edible carrier,
and the like. An "aqueous solvent" means a water-based solvent,
including but not limited to tap water, distilled water, buffers,
salt solutions, and the like.
[0023] The surface-active agent can be an emulsifying, dispersing,
or wetting agent of ionic or nonionic type or a mixture of such
surface-active agents. The presence of at least one surface-active
agent is generally indispensable when the active material and/or
the inert vehicle is /are not soluble in water and the carrier
agent for application is water.
[0024] These compositions can also contain any kind of other
ingredients such as, for example, protective colloids, adhesives,
binding agents, chelating agents, thickening agents, thixotropic
agents, penetrating agents, stabilizing agents, sequestering agents
and the like. The compositions used in the method of the present
invention may also contain other additives depending on the
intended use for the composition. For example, the compositions may
contain anti-foam agents, antioxidants, natural or synthetic
seasonings and/or flavors, dyes and/or colorants, vitamins,
minerals, nutrients, enzymes, insecticides, deodorants, and
mixtures thereof. The amount of such optional additives included in
the composition of the present invention may vary over a wide
range, although amounts of about 0.1 to 10.0 percent of these
compositions are generally satisfactory.
[0025] More generally, the chitosan salts and the essential oils
can be combined with all the solid or liquid additives
corresponding to the conventional formulating techniques.
[0026] As forms of liquid compositions or those intended to
constitute liquid compositions at the time of application,
solutions, in particular water-soluble concentrates, emulsions,
suspension concentrates, aerosols, wettable powders (or powder to
be sprayed), pastes or gels are included. The composition of the
present invention can be presented to the consumer in dry form to
be used after it is wetted with water, i.e., water-activated.
[0027] These compositions can be delivered from for example,
bottles, tubes, pumps, squeeze roamers, bags, wipes, and aerosol
containers as e.g., volatiles, foams, mousses, lathers, wipes, and
dips.
[0028] A composition according to the present invention is most
readily used to treat the surface of solid food products. The
active materials or combinations may be applied to fruits and
vegetables by dipping, spraying, painting, marinating, and/or
wiping the surface. In still other embodiments, the composition may
be applied as a breading, seasoning rub, glaze, colorant mixture,
and the like, the key criteria being that the antimicrobial
composition be available to the surface subject to bacterial or
fungal degradation and/or contamination. In still other
embodiments, the composition may be indirectly placed into contact
with the food surface by applying the composition to food packaging
and thereafter applying the packaging to the food surface. The
optimum amount to be used will depend on the composition of the
particular food product to be treated and the method used for
applying the composition to the food surface, but can be determined
by simple experimentation. It is preferred that the active material
or combination be dissolved or dispersed in a vehicle as defined
above, at concentrations between 10 and 50% solids. When employing
a composition of the invention, the essential ingredients, namely,
the essential oils and/or chitosan salts can advantageously be used
in amounts ranging from about 3000 ppm to about 10 ppm based on
total weight of the food product.
EXAMPLES
[0029] The following examples serve as further description of the
invention and methods for practicing the invention. They are not
intended as being limiting, rather as providing guidelines on how
the invention may be practiced.
Example 1
Bacterial and Fungal Cultures
[0030] E. coli (Strain #139 HB101/p5G6) was grown at 24.degree. C.
for 48 hr in shake-flask cultures of Lennox broth (LB). Bacterial
cells were pelleted by centrifugation in a Sorvall RC-58 centrifuge
(Dupont Instruments, Wilmington, Del.) at 3000 g for 20 min,
resuspended in sterile distilled water, and centrifuged again. The
resulting pellets were dispersed in sterile distilled water and the
cell concentration was adjusted to 10.sup.6 CFU per ml using a
standard optical density (OD) curve with the OD values of 0.1 and 1
representing viable cell counts of 1.times.10.sup.6 and
1.times.10.sup.9, respectively. E. coli 015:H7 and L. monocytogenes
isolates were grown overnight at 37.degree. C. in trypticase soy
broth and brain heart infusion, respectively. The concentration of
cells was adjusted to 10.sup.6 CFU per ml. Botrytis cinerea and
Penicillium expansum were isolated from infected fruit and
maintained on potato dextrose agar (PDA). A spore suspension was
obtained by flooding 2 wk cultures of B. cinerea with sterile
distilled water containing 0.1% (v/v) TWEEN 80. Spore counts were
determined with a hemacytometer and spore concentrations were
adjusted with sterile distilled water to obtain 10.sup.5 spores per
ml.
Example 2
Inhibitory Effect of Essential Oils and Chitosan Salts
[0031] The object of this experiment was to determine the
individual effects of different essential oils and chitosan salts,
and the combined effects of chitosan salts with essential oils on
the growth of the indicator organism E. coli and on spore
germination of B. cinerea. Autoclaved LB broth was amended with
sterile solutions of chitosan salts (chitosan propionate and
chitosan sorbate, Sigma, St. Louis, Mo.), essential oils (tarragon,
basil, peppermint, wintergreen, savory, thyme red, and allspice;
Aroma Vera, Cuber City, Calif.), or combinations of chitosan
propionate and chitosan sorbate with individual essential oils to
obtain a concentration of 0.1% (v/v) and dispensed into sterile
test tubes. Tubes of LB amended with different treatments were
inoculated either with 10.sup.6 CFU per ml of E. coli cells or 500
spores of B. cinerea and incubated on a rotary shaker at 24.degree.
C. for 24 hr. For each microorganism, four replicate tubes of each
treatment were used; each experiment was repeated twice. Botrytis
spore germination was determined microscopically. The viable
bacterial cell number was counted by surface plating serially
diluted samples in triplicate on LB agar medium. Plates were
incubated at 24.degree. C. and colonies were counted at 48 hr.
[0032] Among seven essential oils that were tested for their
antimicrobial activity against both B. cinerea and E. coli, savory,
thyme red, and allspice provided the most effective control of both
B. cinerea and E. coli. These three completely inhibited spore
germination of B. cinerea and substantially reduced the growth of
E. Coli (Table 1).
1TABLE 1 Effect of essential oils on spore germination of Botrytis
cinerea and growth of Escherichia coli after 48 hr of incubation at
24.degree. C. % INHIBITION Essential Oil B. cinerea E. coli Control
0 0 Tarragon 0 5 Basil 0 5 Peppermint 100 9 Wintergreen 100 24
Savory 100 80 Thyme Red 100 80 Allspice 100 84
[0033] The effect of time of exposure on the biostatic or biocidal
activity of the most effective essential oils and combinations of
chitosan salts with essential oils was also assessed. Sterile 0.1 %
solutions of chitosan salts (chitosan propionate and chitosan
sorbate), essential oils (cinnamon, savory, thyme red, and
allspice), or combinations of chitosan salts with individual
essential oils were supplemented with 0.1% of autoclaved LB for E.
coli or 0.1% autoclaved PDB for B. cinerea and dispensed into
sterile 10 ml test tubes.
[0034] Test tube cultures were inoculated either with 10.sup.6 CFU
per ml of E. coli cells or 500 spores of B. cinerea and incubated
on a rotary shaker at 24.degree. C. An individual test tube served
as one replicate and four replicates were sampled after one and
four hr of incubation from each treatment for each microorganism.
Botrytis spore germination and the viability of bacterial cells
were determined as described above.
[0035] In tests of the various essential oil/chitosan salt
combinations against spore germination of B. cinerea and growth of
E. coli, all four essential oil/chitosan salt combinations
completely inhibited spore germination of B. cinerea and growth of
E. coli (Table 2).
2TABLE 2 Biocidal activity of essential oils and different
combinations of natural compounds on spore germination of B.
cinerea and growth of E. coli after 1 and 4 hr. Inhibition (%) Cell
Counts (CFU).sup.a B. cinerea E. coli Treatments 1 hr 4 hr 1 hr 4
hr Control 0 0 TNTC.sup.b TNTC Chitosan sorbate 0 0 544 181
Chitosan propionate 0 0 527 191 Cinnamon 0 100 >600 >600
Savory 0 0 >600 >600 Allspice 0 100 >600 >600 Chitosan
sorbate + Cinnamon 100 100 0 0 Chitosan sorbate + Allspice 100 100
0 0 Chitosan propionate + Savory 100 100 0 0 Chitosan propionate +
Red Thyme 100 100 0 0 .sup.aNumber of colony forming units (CFU) in
100 .mu.l sample. .sup.bTNTC = Too Numerous To Count.
Example 3
Inhibitory Effect of Essential Oils, Chitosan Salts, and
Combinations of Essential Oils and Chitosan Salts
[0036] The individual effects of various essential oils and
chitosan salts, and the combined effects of essential oils and
chitosan salts on the growth of the indicator organism E. coli and
on the postharvest pathogen B. cinerea were determined. To measure
the effects of the various treatments on spore germination of B.
cinerea and growth of the E. coli, the essential oils: bay,
cinnamon, savory, thyme red, allspice, birch, cloves, carvacrol,
and hinokitiol (Aroma Vera, Cuber City, Calif.) and MMW chitosan in
acetic, propionic, and sorbic acids were combined together with B.
cinerea or E. coli to yield final concentrations of 0.1 to 0.025%
for the essential oils and 0.1 to 0.0016% for the chitosan salts.
For the assays, 500 spores of B. cinerea were added to each
treatment in microtiter dishes or three ml of a 2X concentration of
E. coli (i.e., 2.times.10.sup.6 CFU/ml) were combined with three ml
of a 2X concentration of treatment in a 15 ml centrifuge tube,
agitated overnight, and plated after 24 hr onto LB agar plates (100
.mu.l suspension/plate). The surfactant (Triton.times.100) was
present at a final concentration of 0.04%. Similarly, for
experiments measuring synergy, combinations of individual essential
oils (at non-inhibitory concentrations) and chitosan acetate,
chitosan propionate, or chitosan sorbate (at non-inhibitory
concentrations) were combined with B. cinerea or E. coli, as
described above. Four replicate tubes of each treatment were used;
each experiment was repeated twice. The viable bacterial cell
number was counted by surface plating serially diluted samples in
triplicate on LB agar medium. Plates were incubated at 24.degree.
C. and colonies were counted at 48 hr. Botrytis spore germination
was determined microscopically.
[0037] Effects on E. coli Growth:
[0038] Savory, thyme red, and carvacol, tested individually, were
the most effective inhibitors of E. coli growth; each, alone, was
inhibitory at 0.05% (Table 3). Cinnamon and hinokitiol reduced E.
coli growth at 0.075%; no effects were seen at 0.05% or lower. Bay,
cloves, allspice, and birch oil were the least effective
inhibitors; they only inhibited at the final concentration of 0.1%.
No effects were observed at 0.075% or lower.
3TABLE 3 Effect of Concentration of Essential Oil on Growth of E.
coli (CFU.sup.a) Concentration (% v/v) Essential Oil 0.1 0.075 0.05
0.025 Bay 13.5 TNTC TNTC TNTC Cinnamon 0 61.5 TNTC TNTC Cloves 0
TNTC TNTC TNTC Allspice 0 TNTC TNTC TNTC Thyme Red 0 4.5 0.5 TNTC
Savory 11.5 0 6.0 TNTC Birch 0 TNTC TNTC TNTC Carvacrol 0 0 0 TNTC
Hinokitiol 0 812 TNTC TNTC .sup.aNumber of colony forming units
(CFU) in 100 .mu.l sample. .sup.bTNTC = Too Numerous To Count.
[0039] All chitosan salts were effective inhibitors of E. coli
growth at concentrations of 0.1%-0.0063%, but no effect was seen
with concentrations of 0.0032% or lower (Table 4).
4TABLE 4 Effect of Concentration of Chitosan Salts on Growth of E.
coli (CFU.sup.a) Concentration (% v/v) 0.1 0.05 0.025 0.0125 0.0063
0.0032 0.0016 Chitosan- 0 0 1 0 0 TNTC TNTC acetate Chitosan- 0 0.5
0.5 0 26 TNTC TNTC propionate Chitosan- 0 0 0 0 4.5 TNTC TNTC
sorbate .sup.aNumber of colony forming units (CFU) in 100 .mu.l
sample. .sup.bTNTC = Too Numerous To Count.
[0040] Essential oils and chitosan salts, each at concentrations
shown to be non-inhibitory in Tables 3 and 4, were combined with E.
coli as described above and their effectiveness at inhibiting the
growth of E. coli was measured. All combinations inhibited E. coli
growth (Table 5). The individual essential oils and chitosan salts
acted synergistically in combination; each inhibited in combination
at concentrations where they were not individually inhibitory.
Those essential oils that were found to be the least effective
inhibitors of E. coli growth, as shown in Table 3, were effective
inhibitors when tested together with chitosan salts.
5TABLE 5 Effect of Synergistic Combinations of Essential Oils and
Chitosan Salts on Growth of E. coli (CFU.sup.a) Concentration (%
v/v) Chitosan Chitosan Chitosan acetate propionate sorbate Conc.
(%) 0.0032 0.0032 0.0032 Water Water TNTC TNTC TNTC TNTC Bay 0.075
75 180 479 TNTC Cinnamon 0.050 401 582 793 TNTC Savory 0.025 1803
691 1614 TNTC Thyme Red 0.025 1044 115 1328 TNTC Allspice 0.075 0
20 32 TNTC Birch 0.075 640 1621 2713 TNTC Cloves 0.075 34 83 193
1212 Carvacrol 0.025 0 0 2 1430 Hinokitiol 0.075 102 56 60 1108
.sup.aNumber of colony forming units (CFU) in 100 .mu.l sample.
.sup.bTNTC = Too Numerous To Count.
[0041] Spore germination of B. cinerea:
[0042] Similar results were observed when spore germination of B.
cinerea was measured (Tables 6, 7, and 8). The individual essential
oils: bay, cinnamon, allspice and cloves were inhibitory at 0.05%;
savory and thyme red were inhibitory only at 0.1 % (Table 6). Thus,
savory and thyme red, the most effective inhibitors of bacterial
(E. Coli) growth were not as effective in inhibiting spore
germination of the fungus, B. cinerea; bay, cinnamon, allspice and
cloves were more effective.
6TABLE 6 Effect of Concentration of Essential Oils on Spore
Germination of B. cinerea. Percent Inhibition of Spore Germination
Concentration (% v/v) 0.1 0.09 0.08 0.07 0.06 0.05 0.04 0.03 0.02
0.01 Bay 100 100 100 100 100 100 0 0 0 0 Cinnamon 100 100 100 100
100 100 0 0 0 0 Savory 100 0 0 0 0 0 0 0 0 0 Thyme Red 100 0 0 0 0
0 0 0 0 0 Allspice 100 100 100 100 100 100 0 0 0 0 Cloves 100 100
100 100 100 100 100 0 0 0
[0043] Chitosan-sorbate alone was effective in completely
inhibiting B. cinerea spore germination at concentrations of 0.1%
to 0.0175% (Table 7). Complete inhibition of spore germination of
B. cinerea was obtained with chitosan-acetate and
chitosan-propionate at 0.1% and 0.08%.
7TABLE 7 Effect of Concentrations of Chitosan Salts on Spore
Germination of B. cinerea. Percent Inhibition of Spore Germination
Concentration (% v/v) 0.1 0.08 0.06 0.04 0.02 0.0175 0.015 0.0125
Chitosan- 100 100 0 0 0 0 0 0 acetate Chitosan- 100 100 0 0 0 0 0 0
propionate Chitosan- 100 100 100 100 100 100 0 0 sorbate
[0044] Essential oils and chitosan salts, each at concentrations
shown to be non-inhibitory in Tables 6 and 7, were combined with B.
cinerea, as described above, and their effectiveness at inhibiting
spore germination of B. cinerea was measured. All combinations of
essential oils and chitosan salts, at concentrations where they
were not individually inhibitory, showed a synergistic effect and
completely inhibited B. cinerea spore germination (Table 8). Those
essential oils that were found to be the least and the most
effective inhibitors of B. cinerea spore germination (Table 6) were
equally effective when tested together with chitosan salts even
though both the essential oil and the chitosan salt were present at
concentrations where no inhibition had previously been observed
(Tables 6 and 7).
8TABLE 8 Effect of Synergistic Combinations of Essential Oils and
Chitosan Salts on Spore Germination of B. cinerea. Percent
Inhibition of Spore Germination Concentration (% v/v) Chitosan
Chitosan Chitosan Conc. acetate propionate sorbate % (v/v) 0.02
0.02 0.006 Water Bay 0.013 100 100 100 0 Cinnamon 0.013 100 100 100
0 Savory 0.03 100 100 100 0 Thyme Red 0.03 100 100 100 0 Allspice
0.02 100 100 100 0 Cloves 0.01 100 100 100 0 Hinokitiol 0.067 100
100 100 0
Example 4
Effect of Combinations of Chitosan Salts and Essential Oils on E.
coli 015:H7 and L. monocytogenes.
[0045] To determine whether the combinations of essential oils and
chitosan salts that were shown in Examples 1-3 to be effective
inhibitors of growth of the non-pathogenic indicator strain of E.
coli would also effectively inhibit pathogenic bacteria, the most
promising combinations were tested at the USDA ARS Eastern Regional
Research Center (Wyndmoor, Pa.) for their effectiveness in
inhibiting the growth of the pathogenic bacteria L. monocytogenes
and E. coli 01 5:H7, a strain of E. coli pathogenic to humans.
Autoclaved LB was amended with a sterile solution of the
combination of chitosan-sorbate with cinnamon oil, chitosan-sorbate
with allspice, chitosan-propionate with red thyme, or
chitosan-sorbate with savory to obtain a final concentration of 0.1
% (v/v). Test tube cultures were inoculated with 10.sup.6 CFU per
ml of E. coli 0157:H7 or L. monocytogenes and incubated on a rotary
shaker at 240 C. An individual test tube served as one replicate
and four replicates were sampled after 0, 1, 2, and 24 hr of
incubation from each treatment for each bacteria. The viable
bacterial cell number was counted by surface plating serially
diluted samples containing E. coli 015:H7 and L. monocytogenes in
triplicate on LB agar medium and lithium
chloride-phenylethanol-moxalactan agar, respectively. Plates were
incubated at 24.degree. C. and colonies were counted after 48
hr.
[0046] From these tests, it is apparent that all four combinations
of the essential oils and chitosan salts were effective in
inhibiting the growth of E. coli strain 01 57:H7 and L.
monocytogenes (Table 9).
9TABLE 9 Biocidal activity of different combinations of natural
compounds and essential oils on growth of E. coli 0157:H7 and L.
monocytogenes cells after 0, 1, 4, and 24 hr. Bacterial Cell Counts
(Log CFU).sup.a E. coli 0157:H7 L. monocytogenes TREATMENTS 0 hr 1
hr 4 hr 24 hr 0 hr 1 hr 4 hr 24 hr Control 6 6 6 6 6 6 6 6 Chitosan
sorbate + Cinnamon 6 6 6 0 3 0 0 0 Chitosan sorbate + Allspice 6 6
6 0 3 0 0 0 Chitosan propionate + Red Thyme 6 6 0 0 0 0 0 0
Chitosan propionate + Savory 0 0 0 0 0 0 0 0 .sup.aNumber of Colony
Forming Units (CFU) in 100 ml sample expressed in Log CFU/ml.
Example 5
Effect of Essential Oils and Chitosan Salts on Contamination of
Apple Disk with E. coli.
[0047] Experiments were conducted to determine whether cinnamon,
allspice, savory, chitosan sorbate, and chitosan propionate and/or
their combinations could protect fruit surfaces against
colonization by E. coli and whether E. coli could be eradicated
once established on fruit surfaces with these treatments. Tree-ripe
apples (Malus domestica Borkh) cultivar `Red delicious` were
hand-picked at harvest maturity at the Appalachian Fruit Research
Station, Kearneysville, W. Va. Fruit were sorted to remove any with
apparent injuries or infections and stored at 4.degree. C. under
refrigeration before being used in the biocontrol tests. Apple
disks (10 mm) were excised from selected Red delicious apples using
a cork borer. Apple disks were treated by immersion for 90 min in a
0.1% solution of various essential oils and/or their combinations
with 0.1 % chitosan salts. Disks were either treated with the
different combinations of natural compounds and then inoculated
with E. coli by soaking apple disks in a solution of E. coli for 90
min or inoculated with E. coli and incubated at 24.degree. C. for
24 hr. From each treatment four disks were selected randomly,
individually homogenized in 5 ml of sterile water, vortexed, and
dilution plated in triplicate on a LB agar medium. Plates were
incubated at 24.degree. C. and colonies were counted after 48
hr.
[0048] Chitosan sorbate and chitosan propionate in combination with
essential oils of cinnamon, allspice, and savory completely
protected apple disks against colonization by E. coli and
completely eradicated established E. coli growth (Table 10).
10TABLE 10 Protectant and Eradicant Effects of Natural Compounds on
Growth of E. coli on Apple Disks. Protectant Activity.sup.a
Eradicant Activity.sup.b TREATMENTS.sup.e E. coli (CFU).sup.c E.
coli (CFU) Control .sup. TNTC.sup.d TNTC Sorbate TNTC TNTC
Propionate TNTC TNTC Chitosan sorbate 133 248 Chitosan propionate
>1800 >1800 Cinnamon 43 450 Allspice >1800 548 Savory 41
54 Chitosan sorbate + Cinnamon 0 0 Chitosan sorbate + Allspice 0 0
Chitosan propionate + Savory 0 0 .sup.aApple disks were treated
with the different combinations and thereafter immersed in 3 ml
aqueous solution containing 10.sup.6 CFU per ml of E. coli for 90
min. .sup.bApple disks were immersed in 3 ml aqueous solution
containing 10.sup.6 CFU per ml of E. coli for 90 min, drained, and
treated with the different combinations. .sup.cNumber of colony
forming units (CFU) in 100 ml sample. .sup.dTNTC = Too Numerous To
Count. .sup.eAll essential oils and chitosan salts were tested at
0.1% (v/v).
[0049] All publications and patents mentioned in this specification
are herein incorporated by reference to the same extent as if each
individual publication or patent was specifically and individually
indicated to be incorporated by reference.
[0050] It is understood that the foregoing detailed description is
given merely by way of illustration and that modifications and
variations may be made therein without departing from the spirit
and scope of the invention.
* * * * *